1. Field of the Invention
The present invention in the field of biochemistry and medicine is directed to peptides designated PP-1 and PP-2, and functional variants thereof, that prevent apoptosis of lung epithelial cells in vitro and in vivo, and prevent or treat pulmonary fibrosis, making them useful for treating acute lung inflammation and its sequelae, primarily pulmonary fibrosis.
2. Description of the Background Art                Throughout the disclosure, references are cited either by a number(s) in parentheses, referring to the Documents list at the end, or by insertion of the cite into the text.        
Expression of urokinase plasminogen activator (uPA) the uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) and p53 by lung epithelial cells (“LEC”) independently influence a broad range of processes implicated in the pathogenesis of acute lung injury (“ALI”) and its repair (1-5). These processes include cellular proteolysis, adhesion, migration (6), and cellular viability (5).
The present inventors and colleagues recently described extensive cross-talk between the uPA/uPAR/PAI-1 system, p53, and the viability of LECs (5, 7-9). They discovered that uPA-mediated signaling regulates expression of p53 by LECs, which in turn regulates uPA and uPAR or reciprocally promotes expression of the pro-apoptotic effector; PAI-1 (10-12). While these interactions comprise a new paradigm, their role in the pathogenesis of lung injury and pulmonary fibrosis is not yet fully elucidated. This deficiency in the prior art is addressed in part by the present invention.
Lung expression of uPA, uPAR and PAI-1 is of particular importance in the control of proteolysis, fibroblast viability and lung remodeling in Acute Respiratory Distress Syndrome (ARDS) and other forms of ALI (13-20), and interstitial lung diseases (6,13-29). uPA is mitogenic for several cell lines as well as LECs (5, 30-39). The present inventors' group (2-4) and others (40-42) recently showed that uPA regulates PA activity of LECs by inducing its own expression as well as uPAR and PAI-1. These responses occurred as a result of stabilization of the respective mRNAs (2-4). Further studies showed that uPA also regulates epithelial cell apoptosis/survival through regulation of p53 (5) which controls reciprocal expression of uPA (12), its receptor uPAR (10) and its major inhibitor PAI-1 (11) at the posttranscriptional level and involves a novel cell surface signaling interaction between uPA, uPAR, caveolin-1 (“Cav-1”) and β1-integrin (5). Based on the appreciation of the foregoing, the present inventors conceived of new compositions and methods for treating ALI and its consequent remodeling reactions.
uPA and PAI-1 Expression by LECs.
Plasminogen present in plasma or extravascular fluids can be converted to the active endopeptidase plasmin by uPA or tissue-type PA (tPA) (1, 32). While tPA is mainly responsible for intravascular thrombolysis, uPA is involved in extravascular proteolysis and tissue remodeling (47). Two endogenous PA inhibitors, PAI-1 and PAI-2, are produced in epithelial cells, endothelial cells and platelets and (1,4,32,46-51). PAI-1 is primarily responsible for the fibrinolytic defect in bronchoalveolar lavage (BAL) fluids of ARDS patients (6, 13-23). In concert with other resident lung cells, LECs contribute to the derangements seen in these fluids. uPAR and uPA allow proximate activation of plasminogen at the surface of the lung epithelium. This proteolytic system operates in the setting of lung injury and remodeling. The discoveries of the present inventors shed new light on the pathogenesis of both processes (7,15, 19).
uPAR (Vassalli et al., J. Cell. Biol., 100:86, 1985; Ploug et al, J. Biol. Chem., 273:13933, 1998) is a highly glycosylated, glycosyl-phosphoinositol (GPI)-linked receptor implicated in uPA-mediated cellular signaling (2, 52), adhesion (6), proteolysis, differentiation (1, 32, 53) and proliferation (5, 30-33). uPAR also congregates at the leading edge of migrating cells, facilitating cell movement (53-54). uPAR has three extracellular domains (D1-D3) and binds uPA with high affinity via its receptor-binding domain, D1 in the N-terminal portion of the molecule. The functions of D2 and D3 have not been completely elucidated. uPAR binds active 54 kDa uPA, not the low molecular weight (33 kDa) form of active u-PA (Vassalli et al., supra; Cubeilis et al., J. Biol. Chem., 261:15819-22, 1986). Binding to the receptor does not require the catalytic site of uPA and the binding determinant is in the N-terminal part of the enzyme which (in the primary structure) is remote from the catalytic site. The receptor binding domain is in the 15 kDa N terminal fragment (“ATF”, residues 1-135) uPA, more precisely within the Cys-rich region, termed the growth factor region due to homology to the receptor-binding part of epidermal growth factor (EGF). Amino acid residues 12-32 of uPA appear to be critical for binding (Appella et al., J. Biol. Chem., 262:4437-40, 1987).
LECs synthesize and secrete a 55 kDa proenzyme, the single chain form of uPA (or scuPA), which is activated by plasmin and other proteases. LECs also synthesize and express uPAR. LECs express PAI-1 (4,50,55-56), which inhibits uPA activity and promotes cycling of tripartite uPA/uPAR/PAI-1 complexes from the cell surface. Expression of uPA, uPAR and PAI-1 in these cells is augmented by proinflammatory stimuli or inhaled particulates (57-58). Understanding the regulatory interactions of these molecules is currently limited. The discoveries disclosed herein describe new pathways by which these molecules are regulated at the level of mRNA stability. See FIG. 1.
Derangements of Fibrinolysis in Pathogenesis of Human ALI and Pulmonary Fibrosis
Extravascular fibrin promotes the inflammatory response and fibrotic repair after injury (15,18-19). Plasmin facilitates remodeling of the fibrin neomatrix (19). Plasmin generates peptides that injure endothelial cells and induce microvascular leakage (59-60). Plasmin degrades extracellular matrix (ECM) proteins such as laminin and fibronectin and facilitates destruction of collagen and elastin by activating latent matrix metalloproteinases. Thus, successful repair of injured lung parenchyma requires precise balance of plasmin activity.
A deficiency of alveolar plasmin activity characterizes acute and chronic lung injuries. Human BAL fluid normally exhibits high levels of uPA activity, but uPA-dependent fibrinolytic activity is reduced in patients with idiopathic pulmonary fibrosis (IPF), sarcoidosis, ARDS, or severe pneumonias (14-16, 17, 19, 21-23). This defect is mainly attributable to local overexpression of PAI-1 (13-15,28-29). In ALI, uPA is primarily complexed with PAI-1, which is internalized in complex with uPAR and is relatively unavailable to affect local proteolysis or otherwise participate in normal LEC signaling interactions. These conditions perpetuate florid extravascular fibrin in ARDS and other types of lung injury (14-26). Collectively, these observations implicate uPA, uPAR and PAI-1 in the pathogenesis and repair of acute lung injury.
p53 and the Fibrinolytic System in Fibrotic Repair after ALI
Bleomycin (“bleo”), a potent chemotherapeutic agent, causes fibrotic lung disease in humans, rats and mice (25-27,43-45,61-66) and induces acute lung injury before the onset of fibrosis (67-70). Bleo's cytotoxic effect is believed to involve binding to and cleaving DNA (70-74) as a necessary step in development of pulmonary fibrosis (71-72). Bleomycin-induced DNA damage in the lung leads to increased intrapulmonary expression of p53 (69). Cells with elevated p53 typically arrest in the G1 phase and either undergo DNA repair or apoptosis. Increased bleo-induced lung injury occurs with suppression of p53 (75). Complete deficiency of p53 would be deleterious, permitting the persistence of severely damaged LECs and other damaged lung resident cells.
Intratracheal instillation of bleo likewise augments alveolar expression of PAI-1 (25-27,61-64) and p53 (69) and studies of transgenic animals also support a critical role for uPA and its inhibition by PAI-1 in the pathogenesis of pulmonary fibrosis (26-27). PAI-1 deficiency, exogenous uPA and induction of lung-specific uPA protect mice from bleo-induced lung injury (62-64).
Based on these observations, the present inventors conceived that interactions between uPA, p53 and other key components of the fibrinolytic system critically influence outcomes in lung inflammation and repair, and conceived of the therapeutic peptides described herein.
LEC Apoptosis in the Pathogenesis of Lung Inflammation and Pulmonary Fibrosis:
Lung diseases such as ARDS, IPF and other interstitial lung diseases are characterized by LEC apoptosis and progressive fibrosis (18). In asthma and chronic obstructive pulmonary disease (COPD), fibrotic changes occur with apoptosis of the airway epithelium and within subepithelial tissues of the conducting airways (28-29). In all these diseases, remodeling of the lung matrix and LEC apoptosis appear to be mechanistically linked (13,18). Recent reviews suggest a close relationship between uPA-uPAR-mediated matrix remodeling, cellular viability and LEC proliferation (18,76). However, up until the present invention, there was a paucity of evidence directly linking the coordinate control of LEC viability to interactions of uPA with p53.
The present inventors' group recently found that uPA regulates LEC apoptosis and proliferation through elaboration of p53 in a bi-phasic, dose-dependent manner (5).
uPA interacts with uPAR to promote local proteolysis as well as cell proliferation and migration (1-2,5,31,53-54), which are implicated in the pathogenesis of lung inflammation and remodeling. The present inventors' group found that uPA enhanced uPA protein and mRNA expression in human Beas2B cells and primary human small airway LECs (3). The induction was mediated through uPAR. uPA-induced uPA expression involved stabilization of uPA mRNA. Autoinduction of uPA by exposure of LECs to uPA is a newly defined pathway by which this protease can influence expression of local fibrinolytic activity and other uPA-dependent cellular signaling responses germane to lung inflammation (77).
The present inventors' group also found that uPA enhanced uPAR expression and 125I-uPA binding in Beas2B and primary human small airway LECs (2). Induction of uPAR by uPA likewise involved uPAR mRNA stabilization (30). This induction represents a novel pathway by which LECs regulate uPAR-dependent cellular responses that contribute to remodeling in lung injury. Induction of both uPA and uPAR by uPA was blocked by a tyrosine kinase inhibitor and potentiated by prevention of dephosphorylation (2-3,30).
Most recently, the present inventors' group found that uPA enhanced PAI-1 protein and mRNA expression in Beas2B and human LECs. Similar to induction of uPA or uPAR expression, uPA-mediated induction of PAI-1 involved posttranscriptional stabilization of PAI-1 mRNA. Induction of PAI-1 by exposure of LECs to uPA is a newly recognized pathway by which PAI-1 could regulate local fibrinolysis and uPA-dependent cellular responses in the setting of lung injury. The regulation of PAI-1, uPA and uPAR by uPA exposure to LECs represents a newly recognized regulatory mechanism that operates at the level of message stability.
p53 Regulates Expression of uPA, uPAR and PAI-1
The present inventors noted that p53 regulated uPA expression by direct interaction with uPA mRNA. Inhibition of p53 expression by RNA silencing enhanced basal and uPA-mediated uPA protein and mRNA expression with mRNA stabilization. Purified p53 bound to a 35 nucleotide uPA mRNA 3′UTR in a sequence-specific manner, which confirmed a new role for p53 as a uPA mRNA binding protein that reduces its stability and thereby reduces cellular uPA expression (12). The present inventors and colleagues recently reported that p53−/− (H1299) cells expressed robust levels of cell surface uPAR and uPAR mRNA (10). Expression of p53 protein in p53−/− cells suppressed basal and uPA-induced cell surface uPAR protein and increased uPAR mRNA degradation. uPA protein and mRNA were decreased in p53 deficient cells, and introduction of wild-type p53 increased PAI-1 protein and mRNA levels.
These observations demonstrated a novel role for p53 as an mRNA binding protein that increased PAI-1 while decreasing uPAR and uPA expression in human LECs, demonstrating a novel forward feedback system in which p53 regulates expression of key components of the fibrinolytic system by direct binding to specific sequences of the uPA, uPAR and PAI-1 3′UTR, respectively. The present inventors conceived of an important role for these interactions in the regulation of the apoptosis or viability of LECs disclosed herein.
β1-Integrin Signaling in uPA Induction of uPA and uPAR with Concurrent Suppression of PAI-1 and p53: Protection against LEC Apoptosis
The present inventors' group recently showed that pretreatment of cells with anti-β1-integrin antibody blocked uPA-induced p53 expression (5). β1-integrin is associated with uPAR at the LEC surface (78-79), and Cav-1 co-precipitates with uPAR/β1-integrin complexes indicating cross-interaction (80-81). Description of Cav-1, and its scaffolding domain (CSD) are described below.
Anti-β1-integrin antibody activated β1-integrin in LECs by clustering of signaling intermediates, mimicking the effects of relatively high-concentrations (>10 nM) of uPA. Put another way, uPA concentrations >10 nM and activation of cell surface β1-integrin stimulated uPA and uPAR expression while blocking expression of PAI-1 and p53: the net result is protection of LECs from apoptosis. These uPA concentrations are consistent with those used therapeutically (82-88) and may be present in plasma or extravascular fluids in pathophysiologic conditions including sepsis or pneumonia (89).
The present inventors and colleagues found that uPA-mediated Stat3 tyrosine (Y705) phosphorylation (activation) is mediated by interaction of uPA with uPAR (31). uPA also increased uPAR association with β-integrin (89-90) and EGF receptor (EGFR), and directly bound to GP130 in LECs (91). GP130 and EGFR are both receptors that induce Stat3 activation (92-93).
A variety of cells may be stimulated to express uPA during ALI or its resolution, and uPAR and/or other receptors could localize relatively high concentrations of uPA at the cell surface during ALI resolution. The present inventors have conceived that during early ALI, uPA is largely bound by excess PAI-1, undergoing inactivation and accelerated recycling (36,46). As uPA has blocks bleo-induced fibrosis when delivered by aerosol (94), the effects of 20 nM uPA on LECs are likewise relevant in an interventional context.
Caveolin-1 (“Cav-1”) and the Caveolin-1 Scaffolding Domain (CSD)
Caveolin-1, a 22 kDa integral membrane protein, is a principal structural and regulatory component of cell membrane caveolae. Li, S. et al., 1996, J Biol Chem. 271:29182-90, described biological activities of caveolin and the fragment CSD of residues 82-101 corresponding to the cytosolic domain. Caveolin interacted with wild-type c-Src but did not form a stable complex with mutationally activated v-Src. The Src-interacting domain of Cav-1 was within residues 82-101. The CSD functionally suppressed the auto-activation of purified recombinant c-Src tyrosine kinase. This CSD had the following features: (1) was required to form multivalent homo-oligomers of caveolin (2) interacted with G-protein α-subunits and down-regulated their GTPase activity. (3) bound to wild-type H-Ras.(4) it is membrane-proximal, suggesting that it may be involved in other potential protein-protein interactions.
Toya Y et al., Endocrinology, 1998, 139:2025-31, showed that CSD inhibited cardiac adenylyl cyclase more potently than tissue adenylyl cyclases, and suggested use of the peptide as an isoform-selective adenylyl cyclase inhibitor.
Engelman J A et al., J Biol Chem., 1998, 273:20448-55, found that caveolins may function as negative regulators of signal transduction because mutational activation of the c-Neu oncogene down-regulated Cav-1 protein expression in certain cultured cells, and conversely, recombinant overexpression of Cav-1 blocked Neu-mediated signal transduction in vivo. The CSD peptide was sufficient to inhibit Neu autophosphorylation.
Ghosh S et al., J Biol Chem., 1998, 273:22267-71 disclosed that endothelial nitric-oxide synthase (eNOS) is targeted to caveoli through interaction with Cav-1 and that the CSD peptide equivalently inhibited NO synthesis and NADPH oxidation by full-length eNOS. The authors proposed that Cav-1 binding to the eNOS reductase domain compromises its ability to bind calmodulin (CaM) and donate electrons to the eNOS heme, thereby inhibiting NO synthesis.
Yamamoto M et al., Exp Cell Res. 1999, 247:380-8, disclosed that platelet-derived growth factor (PDGF) receptors interacted with Cav-1 in fibroblasts and showed that the CSD peptide from Cav-1 (and from caveolin-3, but not caveolin-2), inhibited PDGF-R autophosphorylation. Cav-1 directly bound to PDGF receptors.
Carman C V et al., J Biol Chem. 1999 274:8858-64, studied regulation of G protein-coupled receptor kinases (GRK's) by caveolin and discovered a specific interaction of GRK2 with the CSD.
Kim J H et al., Biochemistry. 1999, 38:3763-9, reported that Cav-1 interacts with phospholipase D1 (PLD1) via the CSD.
Schlegel, A. et al., J Biol Chem., 1999, 274:22660-7, showed that the CSD was necessary and sufficient for Cav-1-mediated membrane binding in vitro leading to the conclusion that CSD contributes to the membrane attachment of Cav-1.
Nystrom F H et al., Mol Endocrinol. 1999 13:2013-24, disclosed that the CSD binds to a Cav-1 binding motif in the insulin receptor (InsR) kinase domain in vitro. which may differentially modulate insulin signaling to enhance insulin action in cells in which (heterologous) InsRs were expressed but to inhibit insulin effects in fat cells.
Schlegel A et al., J Biol Chem., 2000, 275:21605-17, reported that two separate regions of the Cav-1, one of which was CSD, could anchor green fluorescent protein (GFP) to membranes in vivo. CSD targeted GFP to caveolae, albeit less efficiently than full-length Cav-1
Bucci, M, et al., Nature Med., 2000, 6:1362-7, discloses that Cav-1 regulates signal transduction through binding of the CSD to key signaling molecules. However, it was noted that the physiological importance of Cav-1 in regulating signaling has been difficult to distinguish from its traditional functions in caveolar assembly, transcytosis, and cholesterol transport. A chimeric peptide with a cellular internalization sequence fused to the CSD was efficiently taken up into blood vessels and endothelial cells, and selectively inhibited acetylcholine (Ach)-induced vasodilation and NO production. Systemic administration of the fusion peptide suppressed acute inflammation and vascular leak in mice to the same extent as did a glucocorticoid or an eNOS inhibitor.
Zhu L et al., Am J Physiol Heart Circ Physiol., 2004 286:H195-201, disclosed that facilitated internalization of the CSD attenuated an increase in microvessel permeability mediated platelet-activating factor. Je H D et al., Am J Physiol Heart Circ Physiol. 2004, 286:H91-8, utilized a decoy peptide approach to define the involvement of Cav-1 in PKC-dependent regulation of vascular smooth muscle contractility and found that it has a role in coordinating signaling leading to the regulation of contractility
Gaudreault S B et al., J Biol Chem. 2004, 279:356-62, stated that Cav-1 inhibits the activity of most of its interacting partners. A CSD peptide dramatically inhibited cPLA2 synaptoneurosomes and abolished activation of endogenous PLA2 activity with KCl or melittin. This inhibitory action disclosed as being specific (because a scrambled version of this peptide had no effect). The authors concluded that Cav-1, may interfere with synaptic facilitation and long term potentiation formation in the hippocampus.
Li L et al., Mol Cell Biol., 2003, 23:9389-9404 disclosed that Cav-1 maintains activated Akt in prostate cancer cells through interactions of the CSD with binding sites on serine/threonine protein phosphatases which are inhibited.
Sato Y et al., J Biol Chem. 2004, 279:8827-36, examined the molecular mechanism for inhibition of NO formation by Cav-1 studying the CSD peptide and found that Cav-1 inhibits nNos by a different mechanism than for eNOS and (2) the CSD peptide inhibits interdomain electron transfer from the reductase domain to the oxygenase domain.
Williams T M et al., J Biol Chem. 2004, 279:51630-46, stated that Cav-1 influences the development of human cancers, studying Cav-1 in mammary tumorigenesis and lung metastasis. Complete loss of Cav-1 was required to accelerate tumorigenesis and metastasis. Recombinant expression of Cav-1 in a highly metastatic variant line caused an almost 5-fold reduction in invasion in vitro along with marked reductions in MMP-9 and MMP-2 secretion and enzymatic activity and diminished ERK-½ signaling in response to growth factor stimulation. Delivery of a cell permeable peptide encoding the CSD into Met-1 cells was sufficient to inhibit invasion.
Le Lan C et al., FEBS Lett. 2006 580:5301-5, analyzed the conformational properties of two synthetic peptides, D82-R101 and D82-I109 (both encompassing the CSD (D82-R101)) and reported that a stable helical conformation of the CSD in a membrane mimicking system was only present when the peptide included the L102-I109 hydrophobic stretch, a part of the caveolin intra-membrane domain.
Song L et al., Blood. 2007, 109:1515-23 discussed reduced expression of Cav-1 accompanying the diminished expression of tight junction (TJ)-associated proteins following stimulation of brain microvascular endothelial cells (BMECs) with the chemokine CCL2.
Huang J H et al., J Biol Chem. 2007, 282:6143-52, identified a positive clone in a 12-mer phage peptide library displaying a peptide sequence with high binding to the HIV-1 gp41 core. Cav-1 was said to be a known gp41-binding protein; the 12mer sequence contained a putative gp41-binding motif which also exists in the CSD. The authors suggested this interaction may be essential for fusion pore formation or viral endocytosis and could thereby affect pathogenesis.
Levin A M et al., ACS Chem Biol. 2007, 2:493-500, using double barrel shotgun scanning to dissect binding to two or more targets through combinatorial mutagenesis of one protein that binds to multiple targets, found that the CSD bound to and inhibited both eNOS and protein kinase A (PKA). The CSD oligomerized and deoligomerized to modulate its binding affinity to partner proteins.
In a publication appearing after the making of the present invention, Tourkina E et al., Am J Physiol Lung Cell Mol Physiol. 294:L843-61 (2008 May) (Epub Jan. 18, 2008) stated that lung fibrosis involves overexpression of ECM proteins, primarily collagen, by α-smooth muscle actin (ASMA)-positive cells. Cav-1 was described as a master regulator of collagen expression by cultured lung fibroblasts and of lung fibrosis in vivo. A peptide equivalent to the CSD inhibited collagen and tenascin-C expression by normal lung fibroblasts (NLF) and fibroblasts from scleroderma patients' fibrotic lungs (SLF). CSD peptide inhibited ASMA expression in SLF, but not in NLF. Upregulation of Cav-1 expression by adenovirus resulted in similar inhibition of collagen, tenascin-C, and ASMA expression. The authors suggested that low Cav-1 levels in SLF caused overexpression of collagen, tenascin-C, and ASMA. MEK, ERK, Jun N-terminal kinase, and Akt were hyperactivated in SLF; CSD peptide inhibited their activation and altered their subcellular localization. The paper also disclosed that alterations in signaling molecule activation observed in SLF also occurred in fibrotic lung tissue of scleroderma patients and in mice with bleo-induced lung fibrosis. Systemic administration of CSD peptide to bleo-treated mice blocked epithelial cell apoptosis, inflammatory cell infiltration, changes in tissue morphology, signaling molecule activation and collagen, tenascin-C, and ASMA expression associated with lung fibrosis. The authors stated that “CSD peptide may be a prototype treatment for human lung fibrosis that acts in part by inhibiting expression of ASMA and ECM proteins.”